Plant Cell Reports

, Volume 26, Issue 9, pp 1491–1499 | Cite as

Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya

  • Pious Thomas
  • Sima Kumari
  • Ganiga K. Swarna
  • Devalakere P. Prakash
  • Makki R. Dinesh
Cell Biology and Morphogenesis
First Online:
Received:
Revised:
Accepted:

Abstract

This study establishes the widespread prevalence of fastidious or viable but non-culturable endophytic bacteria in field shoots and in unsuspicious shoot-tip cultures of papaya (Carica papaya L.) against the norm of asepsis in vitro. A total of 150 shoot-tips (approximately 10 mm) were inoculated on MS-based culture medium after surface sterilization of field-derived axillary shoots of cv. Surya during November or January (100 and 50, respectively) when 35–50% cultures showed endophytic microbial growth on culture medium. Indexing of apparently clean cultures using bacteriological media helped in detecting and removing additional 14–17% stocks with covert bacteria during the first two passages. The rest of the stocks stayed consistently index-negative during the first eight subculture cycles, but appeared positive in PCR-screening undertaken thereafter employing universal bacterial 16S rRNA gene primers indicating the association of non-cultivable bacteria. Direct sequencing of the PCR product yielded overlapping nucleotide data signifying mixed template or the presence of diverse endophytic microorganisms. This was confirmed by light microscopy of tissue sap revealing viable bacteria in considerable numbers, which were detected under phase contrast or with negative staining. Planting tissue segments or applying homogenate from these stocks on diverse bacteriological media did not induce the organisms to grow in vitro. The shoot cultures displayed variation in growth and rooting potential, the onus of such variation was solely attributable to the associated microorganisms. The findings were confirmed with additional field shoots and fresh in vitro stocks established subsequently. The observations have implications in micropropagation and all other applications involving plant cell, tissue, organ, and protoplast culture.

Keywords

Bacterial 16S rRNA gene Carica papaya Endophytic bacteria Habituation Microbial contamination Viable but non-culturable bacteria 

Abbreviations

BA

Blood agar

BAP

6-Benzylaminopurine

BIM

Bacteriological indexing medium

BHIA

Brain heart infusion agar

FB

Fastidious bacteria

LA

Luria agar

NA

Nutrient agar

PM

Papaya medium

TSA

Trypticasein soy agar

VA

Viss et al. (1991) agar

VBNC

Viable but non-culturable

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Notes

Acknowledgments

This study was supported by a grant from the Department of Biotechnology, Government of India under the project “Identification of covert endophytic microbes in plant tissue cultures and their management and control”. This publication bears IIHR Contribution No. 83/2006.

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Pious Thomas
    • 1
  • Sima Kumari
    • 1
  • Ganiga K. Swarna
    • 1
  • Devalakere P. Prakash
    • 1
  • Makki R. Dinesh
    • 2
  1. 1.Division of BiotechnologyIndian Institute of Horticultural ResearchBangaloreIndia
  2. 2.Division of Fruit cropsIndian Institute of Horticultural ResearchBangaloreIndia

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